Decreased plasma iron levels in women with hyperglycemia are associated with inflammatory status and involve increased HbA1c, osmotic stability, and volume variability of red blood cells.

BACKGROUND AND AIMS: Iron deficiency tendency in individuals with hyperglycemia influences the HbA1c levels' ability to estimate the stationary blood glucose levels. This study investigated the associations of iron status indicators and HbA1c levels with anthropometric, inflammatory, regulatory, metabolic, and hematologic variables in women with hyperglycemia to most widely characterize this iron deficiency tendency. METHODS: A total of 143 volunteers (68 with normoglycemia and 75 with hyperglycemia) participated in this cross-sectional study. Mann-Whitney test was used to compare groups, and the search for associations between pairs of variables used the Spearman correlation method. RESULTS: In women with hyperglycemia, decreased plasma iron level is associated directly with increased HbA1c (p < 0.001), and these changes are both related to C-reactive protein elevation (p = 0.02 and p < 0.05, respectively) and involve decreased mean hemoglobin concentration (p < 0.01 and p < 0.01), which in turn, involves enhanced osmotic stability (dX) (p < 0.05) and volume variability (RDW) (p < 0.0001) of erythrocytes, as well as decreased indirect bilirubin/total bilirubin ratio (p = 0.04). This indirect bilirubin/total bilirubin decrease, which expresses decreased hemoglobin catabolism, does not seem to be solely a process associated with diminished intracellular concentrations of this protein (p = 0.04) since it is associated with CRP elevation (p = 0.03) and reduced LDL cholesterol (p < 0.0001). CONCLUSIONS: In women with hyperglycemia, decreased plasma iron levels were associated with inflammatory status and involved increased HbA1c, osmotic stability, and volume variability of red blood cells.

Medicines Delivery Proximity Programme (PEMProxi) survey results.

OBJECTIVES: To evaluate patients' and community pharmacies' satisfaction towards the Medicines Delivery Proximity Programme (PEMProxi), including patients' level of satisfaction with pharmaceutical services, medication dispensing and delivery, cost, and time saved, community pharmacies' satisfaction with PEMProxi-related information and procedures, contact with the patients, and timings. Additionally, to evaluate patients' and community pharmacies' perception of PEMProxi's advantages and disadvantages. METHODS: Patients and the community pharmacies included in PEMProxi were contacted by telephone to answer a survey. The patient survey included questions regarding their satisfaction level towards PEMProxi, medication dispensing and delivery, and cost and time saved. Patients were also asked to point out the advantages and disadvantages of PEMProxi and give improvement suggestions. The community pharmacy survey included questions regarding satisfaction with entering PEMProxi, related information, procedures, contact with the patients on PEMProxi, and timings. Programme-related advantages, disadvantages and improvement suggestions were also solicited. RESULTS: A total of 101 patients and 49 community pharmacies were included in the analysis. A large majority of patients were very satisfied with PEMProxi (93.1%). The Programme allowed each patient to save on average 30€ and 8 hours per month. More than 95% of the patients reported medication delivery in adequate conditions (n=100), in a timely manner (n=95) and according to the prescription (n=95). Most pharmacies were satisfied or very satisfied with their participation in PEMProxi (63.3%). Nearly half (53.1%) were surprised by its implementation and 98% would be available to participate with more patients if the Programme was extended. CONCLUSIONS: The PEMProxi programme contributed to more convenient and equitable access to medications by chronic patients, thus avoiding unnecessary trips to the hospital, saving them precious time and money.

First Report of Echinococcus ortleppi in Free-Living Wild Boar (Sus scrofa) from Portugal.

Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors' knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock-wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field.

Hyperkyphosis is not dependent on bone mass and quality in the mouse model of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-ß. Angiotensin II (ang-II) is involved in TGF-ß activity and in bone mass regulation. Inhibition of TGF-ß signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8 ± 6.3 vs. 29.7 ± 7.7 µm), coupled with a reduction in type I (164.1 ± 8.7 vs. 139.0 ± 4.4) and increase in type III (86.5 ± 10.2 vs. 140.4 ± 5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0 ± 420.6 vs. 655.6 ± 239.5 µm2) surrounded by increased area of connective tissue (58.17 ± 6.52 vs. 105.0 ± 44.54 µm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

Red cell distribution width and erythrocyte osmotic stability in type 2 diabetes mellitus.

This study aimed to investigate the relationship between red cell distribution width (RDW) and erythrocyte osmotic stability in non-diabetic and diabetic individuals in both sexes. The study sample (N = 122) was constituted by 53 type 2 diabetics (DM) and 69 non-diabetics (ND), being 21 and 22 men in each group, respectively. The osmotic stability of erythrocytes was obtained by the variation in saline concentration (dX) capable of determining hypoosmotic lysis. Higher RDW values and lower serum iron concentrations were found in the diabetic group when compared to the non-diabetic volunteers. In the group of diabetic women, RDW was positively correlated with the reticulocyte index, and both RDW and dX were negatively correlated with iron, haemoglobin, transferrin saturation index, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration. In all the groups studied, RDW was positively correlated with dX, especially in the diabetic group, where the correlation was the strongest. RDW elevation in both women and men with type 2 diabetes mellitus was associated with decreased serum iron indicators. Furthermore, RDW has a similar meaning to dX, as small erythrocytes have less haemoglobin, resulting in both an increase of RDW and dX.

The impact of intrauterine growth restriction on respiratory outcomes.

BACKGROUND: Intrauterine growth restriction (IUGR) is caused by fetal growth below what is normal for its genetic potential. Recent studies have shown a distinct association between changes in umbilical artery flow in IUGR subjects and an increased risk of respiratory morbidity and consequently, higher mortality. The aim of this study was to find the impact of IUGR on the respiratory outcomes of premature neonates born with less than 32 weeks gestational age. METHODS: This retrospective cohort study targeted infants born with less than 32 weeks of gestation, admitted at NCIU, between January 2010 and December 2016. Each selected IUGR case was matched according to gestational age and sex with an appropriate birthweight newborn at a 1:2 ratio, within a 12-month period. RESULTS: The study involved 126 neonates, 42 with IUGR, and 84 control subjects. IUGR was not identified as a predictor of Bronchopulmonary Dysplasia (BDP) (OR 4.80, 95% CI: 1.14-20.21, P=0.033). Abnormal umbilical artery flow (OR 4.80, 95% CI: 1.14-20.21, P=0.033) and late onset sepsis (OR 3.31, 95% CI: 1.04-10.56, P=0.044) were significantly associated with BDP. CONCLUSIONS: It is essential to recognize changes in the umbilical artery flow, especially in high-risk pregnancies such as IUGR, since these represent an a priori risk marker for the development of BDP. The individual and combined effect of IUGR, alterations on umbilical artery flow and extreme prematurity has not yet been completely clarified on the impact on lung morbidity, requiring a larger number of studies.

PCR Detection of Toxoplasma gondii in European Wild Rabbit (Oryctolagus cuniculus) from Portugal.

Wildlife plays an important role in the epidemiological cycle of Toxoplasma gondii. The European wild rabbit (Oryctolagus cuniculus) can be a source of infection to wild and domestic hosts, including human beings. Additionally, as an herbivorous animal, the European wild rabbit may also be a sentinel of environmental contamination with T. gondii and, consequently, an indicator of the potential transmission of this parasite. The purpose of the present work was to detect T. gondii DNA in European wild rabbit from central Portugal, as well as the possible implications for public health. Heart and diaphragm samples were obtained from 28 rabbits hunted in central Portugal. Nested PCR separately amplified the 5' and 3' ends of the surface antigen 2 (SAG2) gene. T. gondii DNA was detected in 19 out of the 28 sampled animals, resulting in a prevalence of 67.9%. These results show that T. gondii infection occurs in European wild rabbit and therefore may pose a potential risk for humans if consumed as raw or undercooked meat. Measures should be taken in order to prevent infection by this zoonotic parasite and for the conservation of wildlife. To the best of our knowledge, this is the first study performed by means of PCR on T. gondii in European wild rabbit meat samples.

[The Importance of Autopsy in Early Neonatal Death in Portugal]. / A Importância da Autópsia na Morte Neonatal Precoce em Portugal.

INTRODUCTION: The early neonatal period is the most critical for the newborn's life. The autopsy is important to understand the cause of death, and find other diagnoses not clinically identified. However, the rate of neonatal autopsy is declining worldwide. This study aims to characterize early neonatal death and the clinical importance of the autopsy, evaluating the concordance between clinical and pathological diagnosis. MATERIAL AND METHODS: Retrospective study of the clinical records of all neonates admitted to a level III Neonatal Intensive Care unit in Portugal who died during the first week of life in 10 consecutive years (2008 - 2017). In order to classify the concordance found between clinical and pathological diagnoses, the modified Goldman classification was used. RESULTS: During the first week of life, 76 newborns died. The main causes of death were complications related with prematurity and congenital malformations. The autopsy was performed in 50 newborns. Additional findings were found in 62% of the cases, and in 12% findings with important implications for genetic counseling of future pregnancies. There was concordance between the clinical and pathological findings in 38% of cases. DISCUSSION: An autopsy was performed more frequently in newborns with greater gestational age. The number of additional diagnoses found at autopsy, including diagnoses with implications for genetic counseling, confirm the importance of performing them. CONCLUSION: An autopsy should be proposed to all parents after early neonatal death, given its importance in clarifying the cause of death.

Introdução: O período neonatal precoce é o mais crítico para a vida do recém-nascido. A autópsia é importante para compreender acausa de morte e conhecer outros diagnósticos não identificados clinicamente. No entanto, a taxa de autópsia neonatal está a diminuir em todo o mundo. Este estudo pretende caracterizar a morte neonatal precoce e a importância clínica da autópsia, avaliando a concordância entre o diagnóstico clínico e o anatomopatológico.Material e Métodos: Estudo retrospetivo dos processos clínicos de todos recém-nascidos admitidos numa unidade de CuidadosIntensivos Neonatais de nível III em Portugal e que faleceram durante a primeira semana de vida em 10 anos consecutivos (2008 - 2017). Para classificar a concordância encontrada entre os diagnósticos clínicos e anatomopatológicos foi usada a classificação de Goldman modificada.Resultados: Na primeira semana de vida faleceram 76 recém-nascidos. As principais causas de morte foram complicações relacionadas com prematuridade e anomalias congénitas. A autópsia foi realizada em 50 (65,8%) recém-nascidos. Achados adicionais foram encontrados em 62% dos casos, sendo em 12% achados com implicações importantes no aconselhamento genético de futuras gestações. A concordância entre os achados clínicos e anatomopatológicos foi de 38% dos casos.Discussão: A autópsia foi realizada com maior frequência em recém-nascidos com maior idade gestacional. O número de diagnósticos adicionais encontrados na autópsia, incluindo diagnósticos com implicações para aconselhamento genético, confirmam a importância da sua realização.Conclusão: A autópsia deve ser proposta a todos os pais após a morte neonatal precoce, dada a sua importância no esclarecimento da causa de morte.

Overexpression of MIG-6 in the cartilage induces an osteoarthritis-like phenotype in mice.

BACKGROUND: Osteoarthritis (OA) is the most common form of arthritis and characterized by degeneration of the articular cartilage. Mitogen-inducible gene 6 (Mig-6) has been identified as a negative regulator of the epidermal growth factor receptor (EGFR). Cartilage-specific Mig-6 knockout (KO) mice display increased EGFR signaling, an anabolic buildup of the articular cartilage, and formation of chondro-osseous nodules. Since our understanding of the EGFR/Mig-6 network in the cartilage remains incomplete, we characterized mice with cartilage-specific overexpression of Mig-6 in this study. METHODS: Utilizing knee joints from cartilage-specific Mig-6-overexpressing (Mig-6over/over) mice (at multiple time points), we evaluated the articular cartilage using histology, immunohistochemical staining, and semi-quantitative histopathological scoring (OARSI) at multiple ages. MicroCT analysis was employed to examine skeletal morphometry, body composition, and bone mineral density. RESULTS: Our data show that cartilage-specific Mig-6 overexpression did not cause any major developmental abnormalities in the articular cartilage, although Mig-6over/over mice have slightly shorter long bones compared to the control group. Moreover, there was no significant difference in bone mineral density and body composition in any of the groups. However, our results indicate that Mig-6over/over male mice show accelerated cartilage degeneration at 12 and 18 months of age. Immunohistochemistry for SOX9 demonstrated that the number of positively stained cells in Mig-6over/over mice was decreased relative to controls. Immunostaining for MMP13 appeared increased in areas of cartilage degeneration in Mig-6over/over mice. Moreover, staining for phospho-EGFR (Tyr-1173) and lubricin (PRG4) was decreased in the articular cartilage of Mig-6over/over mice. CONCLUSION: Overexpression of Mig-6 in the articular cartilage causes no major developmental phenotype; however, these mice develop earlier OA during aging. These data demonstrate that Mig-6/EGFR pathways are critical for joint homeostasis and might present a promising therapeutic target for OA.

Thyrotoxicosis Involves ß2-Adrenoceptor Signaling to Negatively Affect Microarchitecture and Biomechanical Properties of the Femur.

Background: Thyrotoxicosis increases bone turnover, resulting in net bone loss. Sympathetic nervous system (SNS) activation, via ß2-adrenoceptor (ß2-AR) signaling, also has osteopenic effects. Because thyroid hormones (TH) interact with the SNS to regulate several physiological processes, we hypothesized that this interaction also occurs to regulate bone mass. Previous studies support this hypothesis, as α2-AR knockout (KO) mice are less susceptible to thyrotoxicosis-induced osteopenia. Here, we evaluated whether TH-SNS interactions in bone involve ß2-AR signaling. Methods: Thyrotoxicosis was induced in 120-day-old female and male mice with ß2-AR gene inactivation (ß2-AR-/-) by daily treatment with supraphysiological doses of triiodothyronine (T3) for 12 weeks. The impact of thyrotoxicosis on femoral bone microarchitecture, remodeling, fracture risk, and gene expression of the receptor activator of nuclear factor-kappa-B (RANK)-RANK ligand (RANKL)-osteoprotegerin (OPG) pathway was evaluated. In addition, the effect of the ß2-AR-specific agonist clenbuterol (CL) on cAMP accumulation was determined in osteoblastic (MC3T3-E1) cells treated with T3 and/or 17ß-estradiol (E2). Results: Thyrotoxicosis negatively affected trabecular bone microarchitecture in wild-type (WT) females, but this effect was milder or nonexistent in ß2-AR-/- animals, whereas the opposite was seen in males. T3 treatment increased the femoral RANKL/OPG mRNA ratio and the endosteal perimeter and medullary area of the diaphysis in WT females and males, but not in ß2-AR-/- mice, suggesting that T3 promotes endosteal resorption in cortical bone, in a mechanism that involves ß2-AR signaling. T3 treatment increased endocortical mineral apposition rate only in WT females but not in ß2-AR-/- mice, suggesting that TH also induce bone formation in a ß2-AR signaling-dependent mechanism. T3 treatment decreased femoral resistance to fracture only in WT females, but not in KO mice. E2 and CL similarly increased cAMP accumulation in MC3T3-E1 cells; whereas T3 alone had no effect, but it completely blocked E2-stimulated cAMP accumulation, suggesting that some T3 effects on bone may involve E2/cAMP signaling in osteoblasts. Conclusions: These findings sustain the hypothesis that T3 interacts with the SNS to regulate bone morphophysiology in a ß2-AR signaling-dependent mechanism. The data also reveal sex as an important modifier of skeletal manifestations of thyrotoxicosis, as well as a modifier of the TH-SNS interactions to control bone microarchitecture, remodeling, and resistance to fracture.

Abnormal Thyroid Hormone Status Differentially Affects Bone Mass Accrual and Bone Strength in C3H/HeJ Mice: A Mouse Model of Type I Deiodinase Deficiency.

C3H/HeJ (C3H) mice are deficient of type I deiodinase (D1), an enzyme that activates thyroid hormone (TH), converting thyroxine (T4) to triiodothyronine (T3). Nevertheless, C3H mice present normal serum T3 and a gross euthyroid phenotype. To investigate if a global D1 deficiency interferes in the TH effects on bone, we compared bone growth, bone mass accrual and bone strength of C3H and C57BL/6J (B6) mice under abnormal TH status. Four-week-old female mice of both strains were grouped as Euthyroid, Hypothyroid (pharmacologically-induced), 1xT4 and 10xT4 (hypothyroid animals receiving 1- or 10-fold the physiological dose of T4 /day/16 weeks). Hypothyroidism and TH excess similarly impaired body weight (BW) gain and body growth in both mice strains. In contrast, whereas hypothyroidism only slightly impaired bone mineral density (BMD) accrual in B6 mice, it severely impaired BMD accrual in C3H mice. No differences were observed in serum and bone concentrations of T3 between hypothyroid animals of both strains. Interestingly, treatment with 10xT4 was less deleterious to BMD accrual in C3H than in B6 mice and resulted in less elevated T3 serum levels in B6 than in C3H mice, which is probably explained by the lower D1 activity in C3H mice. In addition, hypothyroidism decreased bone strength only in C3H but not in B6 mice, while TH excess decreased this parameter in both strains. These findings indicate that D1 deficiency contributes to the TH excess-induced differences in bone mass accrual in C3H vs. B6 mice and suggest that deiodinase-unrelated genetic factors might account for the different skeleton responses to hypothyroidism between strains.

Global Disruption of α2A Adrenoceptor Barely Affects Bone Tissue but Minimizes the Detrimental Effects of Thyrotoxicosis on Cortical Bone.

Evidence shows that sympathetic nervous system (SNS) activation inhibits bone formation and activates bone resorption leading to bone loss. Because thyroid hormone (TH) interacts with the SNS to control several physiological processes, we raised the hypothesis that this interaction also controls bone remodeling. We have previously shown that mice with double-gene inactivation of α2A- and -adrenoceptors (α2A/2C-AR-/-) present high bone mass (HBM) phenotype and resistance to thyrotoxicosis-induced osteopenia, which supports a TH-SNS interaction to control bone mass and suggests that it involves α2-AR signaling. Accordingly, we detected expression of α2A-AR, α2B-AR and α2C-AR in the skeleton, and that triiodothyronine (T3) modulates α2C-AR mRNA expression in the bone. Later, we found that mice with single-gene inactivation of α2C-AR (α2C-AR-/-) present low bone mass in the femur and HBM in the vertebra, but that both skeletal sites are resistant to TH-induce osteopenia, showing that the SNS actions occur in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss. To further dissect the specific roles of α2-AR subtypes, in this study, we evaluated the skeletal phenotype of mice with single-gene inactivation of α2A-AR (α2A-AR-/-), and the effect of daily treatment with a supraphysiological dose of T3, for 4 or 12 weeks, on bone microarchitecture and bone resistance to fracture. Micro-computed tomographic (µCT) analysis revealed normal trabecular and cortical bone structure in the femur and vertebra of euthyroid α2A-AR-/- mice. Thyrotoxicosis was more detrimental to femoral trabecular bone in α2A-AR-/- than in WT mice, whereas this bone compartment had been previously shown to present resistance to thyrotoxicosis in α2C-AR-/- mice. Altogether these findings reveal that TH excess depends on α2C-AR signaling to negatively affect femoral trabecular bone. In contrast, thyrotoxicosis was more deleterious to femoral and vertebral cortical bone in WT than in α2A-AR-/- mice, suggesting that α2A-AR signaling contributes to TH actions on cortical bone. These findings further support a TH-SNS interaction to control bone physiology, and suggest that α2A-AR and α2C-AR signaling pathways have key roles in the mechanisms through which thyrotoxicosis promotes its detrimental effects on bone remodeling, structure and resistance to fracture.

Thyroid Hormone and Skeletal Development.

Thyroid hormone (TH) is essential for skeletal development from the late fetal life to the onset of puberty. During this large window of actions, TH has key roles in endochondral and intramembranous ossifications and in the longitudinal bone growth. There is evidence that TH acts directly in skeletal cells but also indirectly, specially via the growth hormone/insulin-like growth factor-1 axis, to control the linear skeletal growth and maturation. The presence of receptors, plasma membrane transporters, and activating and inactivating enzymes of TH in skeletal cells suggests that direct actions of TH in these cells are crucial for skeletal development, which has been confirmed by several in vitro and in vivo studies, including mouse genetic studies, and clinical studies in patients with resistance to thyroid hormone due to dominant-negative mutations in TH receptors. This review examines progress made on understanding the mechanisms by which TH regulates the skeletal development.

Early versus late repair of rotator cuff tears in rats.

BACKGROUND: In the event of a traumatic rotator cuff tear, patients are routinely advised that early surgical intervention produces an optimal repair, despite a lack of direct evidence to support this recommendation. To address this knowledge gap, massive rotator cuff tears in rats were assessed by biomechanical and bone morphometric analyses after early or late repair. METHODS: Combined supraspinatus and infraspinatus tendon tears of the left shoulder were created in 21 adult Wistar rats, which were divided into 2 groups. The tendons of the injured shoulder in the animals in group I were surgically repaired 8 weeks after the injury. Under the same anesthesia, the same injury was created on the right shoulder, which was immediately repaired. The rats from group I were euthanized 8 weeks after the repairs. No repair was performed in the rats from group II, which were euthanized 8 weeks after the injury. Tissues from both groups were harvested and biomechanically tested for supraspinatus tendon and bone morphometry analysis of the humeral head. RESULTS: All biomechanical properties were significantly increased in the early repair group compared with the late repair group. No significant differences were observed in bone morphometry of the humeral head when early and late repair groups were compared. CONCLUSION: Early surgical repair of a massive rotator cuff tear leads to improved biomechanical properties of the tissue after healing. Proximal humerus bone morphometry was unaffected by surgical repair timing.

Analysis of pharmaceutical adulterants in plant food supplements by UHPLC-MS/MS.

A method based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was successfully developed and validated for the analysis of 26 pharmaceutical compounds belonging to different therapeutic classes (anorexics, stimulants, anxiolytics, antidepressants and laxatives), which are all prone to be illegally added into weight-loss plant food supplements (PFS) for their pharmacological activity. Internal standard calibration with six isotopically labelled compounds rendered good linearity in the range of 5 to 1000µg/l, depending on the compound, and good sensitivity with limits of quantification in the range of 0.02-9.80µg/l. Recoveries were assessed for all the 16 samples analysed and were found between 70% and 120% for over 90% of the analytes. The average recovery value was 90.8%, for the different studied matrices (liquids, liquid ampoules, tablets and capsules), with RSD values lower than 10% for all forms. The changes introduced to the QuEChERS procedure maintained the good performance characteristics of the extraction method while preserving the chromatographic system for the introduction of unwanted matrix compounds. Synephrine was the only compound detected and quantified in one sample, but at a very low concentration (768µg/l) and its presence may be due to the plant extracts used in the formulation, as synephrine is known to be a natural constituent of Citrus aurantium amara. Despite none of the 16 evaluated samples were found to be adulterated by the illegal addition of the drugs included in this work, the developed methodology can be very useful for monitoring the adulteration of weight-loss PFS.

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia.

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via ß2-adrenoceptor (ß2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that ß2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (µCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.

Genotyping Characterization of Toxoplasma gondii in Cattle, Sheep, Goats and Swine from the North of Portugal.

BACKGROUND: Epidemiological investigations on Toxoplasma gondii infection have found a significant association between human toxoplasmosis and consumption of raw or undercooked meat. The present study aimed to characterize genotypes of T. gondii in 20 cattle, 40 sheep, 15 goats and 16 pigs from the North of Portugal. METHODS: Nested PCR amplified the surface antigen 2 (SAG2) gene. Sequencing analysis was performed in order to assess the prevalence of SAG2 type strains (I, II and III). RESULTS: Three and 4 strains of SAG2 type II were identified in heart samples of cattle and sheep, respectively. Three SAG2 type II strains were detected in brain, diaphragm and heart of 3 pigs. Three strains detected in heart samples of 3 goats belonged to SAG2 types I or II; with the same result being observed in heart samples from 2 sheep and in 2 brain and 1 heart samples from 3 pigs. CONCLUSION: SAG2 type II has been shown for the first time to infect cattle in North of Portugal. In addition, SAG2 type II has also been confirmed as the predominant strain in sheep and pigs in the same region. This is the first molecular report of T. gondii in goats from Portugal.

Thyroid hormone interacts with the sympathetic nervous system to modulate bone mass and structure in young adult mice.

To investigate whether thyroid hormone (TH) interacts with the sympathetic nervous system (SNS) to modulate bone mass and structure, we studied the effects of daily T3 treatment in a supraphysiological dose for 12 wk on the bone of young adult mice with chronic sympathetic hyperactivity owing to double-gene disruption of adrenoceptors that negatively regulate norepinephrine release, α(2A)-AR, and α(2C)-AR (α(2A/2C)-AR(-/-) mice). As expected, T3 treatment caused a generalized decrease in the areal bone mineral density (aBMD) of WT mice (determined by DEXA), followed by deleterious effects on the trabecular and cortical bone microstructural parameters (determined by µCT) of the femur and vertebra and on the biomechanical properties (maximum load, ultimate load, and stiffness) of the femur. Surprisingly, α(2A/2C)-AR(-/-) mice were resistant to most of these T3-induced negative effects. Interestingly, the mRNA expression of osteoprotegerin, a protein that limits osteoclast activity, was upregulated and downregulated by T3 in the bone of α(2A/2C)-AR(-/-) and WT mice, respectively. ß1-AR mRNA expression and IGF-I serum levels, which exert bone anabolic effects, were increased by T3 treatment only in α(2A/2C)-AR(-/-) mice. As expected, T3 inhibited the cell growth of calvaria-derived osteoblasts isolated from WT mice, but this effect was abolished or reverted in cells isolated from KO mice. Collectively, these findings support the hypothesis of a TH-SNS interaction to control bone mass and structure of young adult mice and suggests that this interaction may involve α2-AR signaling. Finally, the present findings offer new insights into the mechanisms through which TH regulates bone mass, structure, and physiology.

[End of life in neonatology: palliative care integration]. / Fim de vida em neonatologia: integração dos cuidados paliativos.

BACKGROUND: Today the limitation of therapeutic effort and the neonatal palliative care are extremely important and had been increasingly done by several countries. Nevertheless, in Portugal, studies and guidelines regarding end of life attitudes are still a shortage. MATERIAL AND METHODS: We analyzed 49 files in a retrospective review of neonatal deaths between 2010 and 2012; the results were compared with those from a previous study made in the same NICU that included the neonatal deaths of 1992-1995 and 2002-2005. RESULTS: The results show that congenital anomalies were the cause of death for 57.1% newborns, extreme prematurity for 18.4% and 16.3% died with infection. Regarding to the mode of death, 57.1% of newborns died after cardiopulmonary resuscitation failure, 20.4% after withholding treatment and 22.4% of deaths followed therapy withdrawal. Differences were found between the three groups classified according to the 'mode of death', length of stay and parental presence during death. Therapeutic limitation practices and palliative care were reported in 28.6% of the files and a Do-Not-Resuscitate Order was included in 16.3%. The former two increased during the three periods analyzed. The use of a neonatal pain scale, opioids and sedatives administration, parental presence during death, clinical meetings with neonatologists, interdisciplinary meetings and psychological support for parents also increased. CONCLUSION: This study shows a trend towards an increase in therapeutic limitation practices and palliative care in this NICU in the last three decades. However, there is still a lot to do regarding the clinical research, medical education and ethical discussion.

Introdução: A limitação terapêutica e a implementação dos cuidados paliativos em Neonatologia revestem-se de extrema relevância na atualidade e são já uma realidade com tendência crescente em vários países. Não obstante, em Portugal, os estudos, recomendações e protocolos oficiais no que diz respeito às questões em fim de vida são ainda escassos.Material e Métodos: Analisamos retrospetivamente os processos clínicos dos recém-nascidos falecidos na Unidade de Cuidados Intensivos do Centro Hospitalar de São João no período de 2010 a 2012 e comparamos os resultados com os obtidos num estudo realizado na mesma Unidade correspondente aos períodos 1992-1995 e 2002-2005.Resultados: Na população estudada as anomalias congénitas foram a causa de morte em 57,1% casos, a prematuridade extrema em 18,4% casos e a infeção em 16,3%. No que refere ao 'modo de morrer', a paragem cardiorrespiratória irreversível às manobras de reanimação observou-se em 57,1% casos; 20,4% faleceram após abstenção de tratamento e 22,4% após suspensão de tratamento. Verificaram-se diferenças significativas para o 'modo de morrer' e as variáveis tempo de internamento e presença dos pais no momento da morte. Em 28,6% casos houve registo de tomada de decisão pela limitação terapêutica e adoção de cuidados paliativos e em 16,3% decisão de não reanimar. Para as duas últimas registou-se um aumento ao longo das três décadas analisadas. Também se registou uma tendência crescente para a utilização de escala de avaliação de dor neonatal, opióides e sedativos, presença parental no momento da morte, reuniões entre pais e neonatologistas, reuniões interdisciplinares e apoio psicológico aos pais.Conclusão: Este estudo revela uma tendência crescente para a integração de medidas de limitação terapêutica e de cuidados paliativos na UCIN estudada. Contudo, há ainda muito a desenvolver, nomeadamente no que refere à investigação, formação dos profissionais e debate ético.